This proposed project is to address provocative question 2 from Group C: How can the physical properties of tumors, such as the cell's electrical, optical or mechanical properties, be used to provide earlier or more reliable cancer detection, diagnosis, prognosis, or monitoring of drug response or tumor recurrence? We will investigate the optical properties and their associated nanoscale architectural changes in the cell nucleus during carcinogenesis and determine their accuracy in providing earlier and more accurate diagnosis and prognosis of breast cancer. We hypothesize that the nanoscale alterations in nuclear architecture occur early in carcinogenesis and the measurement of easily obtained optical markers of nanoscale changes in nuclear architecture can serve as a cost-effective and accurate tool for earlier and more accurate cancer diagnosis and prognosis. Our group has developed a set of optical microscopy systems that can comprehensively characterize 3D nanoscale alterations in nuclear architecture in carcinogenesis using clinically obtained routine formalin-fixed and paraffin-embedded tissue. Our optical microscopy systems include depth-resolved spatial-domain low-coherence quantitative phase microscopy (depth-resolved SL-QPM) and spectral-encoding of spatial frequency (SESF). We showed that depth-resolved SL-QPM detects structural changes at a sensitivity of 1 nm within a single cell nucleus, while SESF extracts the structural length-scale distribution at an accuracy of ~10-20 nm. Our extensive preliminary data have shown the promise of these optical markers to detect the presence of invasive cancer even from histologically normal cells from multiple tumor types and predict cancer progression risk. Now we propose to use these two optical microscopy systems together with state-of-the-art 3D super-resolution microscopy, to define a set of optical markers and the underlying nanoscale changes in nuclear architecture that are characteristic of each phase of tumorigenesis and identify those that detect premalignant changes. Then we will perform a clinical study to evaluate the accuracy of optical markers of nanoscale changes in nuclear architecture to predict breast cancer progression risk among women with pre-cancerous lesions (e.g., atypical hyperplasia (AH)) and pre-invasive cancer of ductal carcinoma in situ (DCIS) to avoid over- treatment. This project, if successful, will establish the alterations of nanoscale nuclear architecture in carcinogenesis, and have profound impact on both tumor biology research and clinical care. It will build a solid foundation for future use of optical markers of nanoscale changes in nuclear architecture as accurate prognostic markers to predict those women that are likely to progress into invasive cancer.